1. Field of the Invention:
This invention relates to a method for continuously or periodically determining the radioactivity of eluates from a liquid chromatography column and to an apparatus by which this determination may be implemented. More particularly, it relates to a method for adding, quantitatively, small volumes of scintillation cocktail to eluates from liquid chromatography columns, and it is specifically adapted to the determination of the radioactivity in eluates from high pressure liquid chromatography columns. Additionally, this method allows the simultaneous determination of radioactivity and the preservation of aliquots of the eluate for analysis by other means which cannot be performed in the presence of scintillation cocktail solutions.
2. Background of the Invention:
Radioactive isotopes are frequently used in chemical and biochemical laboratory experiments to determine the mechanism of reaction, the rate of reaction, the structure of a particular chemical species, and the identity of physiologically-active cellular receptors. Analytical techniques involving the use of radioisotopes have broad applications in both research and process control. Radioisotopes are particularly useful for the determination of nanogram and smaller amounts of material not normally measurable by other techniques.
The separation of chemical and especially biochemical components in solution by elution from a packed column containing inert adsorbants is a well practiced art. Typically, aliquots of the eluate were collected on the basis of color, absorbance of ultraviolet radiation, or time. When one or more components also contained a radioactive isotope, the aliquots could be further identified on the basis of this radioactivity, typically by mixing the eluate with a known volume of scintillation cocktail and counting the activity in a scintillation counter. The automation of such a process, whereby the ultraviolet absorbance and the radioactivity of the eluates can be determined is particularly advantageous when the elution time is measured in hours or is performed overnight. An automated system for such measurement is described in Schutte (J. Chromatogr., 72 303 (1972)) and a variation is described in U.S. Pat. No. 4,194,117 to Gross, issued Mar. 18, 1980.
High pressure liquid chromatography is an improvement over traditional liquid chromatography characterized by the ability to analyze small volumes of solution at comparatively higher flow rates by the use of a high pressure pump to supply the solvent to the column. Typical analytical high performance liquid chromatography instruments provide for the analysis of microlitre quantities of solution and employ stainless steel capillary tubing and small volume columns. While the automated systems described above are suitable for large volume systems, typically those in which the solutions are delivered via a peristaltic pump through flexible plastic tubing, analytical high pressure liquid chromatographs require finer control of the amount of scintillation cocktail added to the eluate because of the small volume of sample employed. In the analysis of many biological systems, it is frequently necessary to measure, in addition to the above, the sedimentation rate or the isoelectric point of proteins or to purify the material in the eluate for further use. The principle of eluate stream diversion at timed intervals consists of diverting the eluate stream for a time interval T.sub.1, and passing the stream to a collector, such as a fraction collector, for a time interval T.sub.2. The ratio of the mean flow volumes is obtained by averaging the flow rate over an operating cycle T.sub.1 +T.sub.2 is approximated by the ratio of the interval durations of the two phases of the operating cycle, VLZ T.sub.1 /T.sub.2. A device for performing such a timed split is that described in Radiomatric Instruments and Chemical Co. Inc. brochure 120/681. Under such conditions, the volume of scintillation cocktail required for reproduceable results becomes quite small and must be delivered at high pressure. Continous flow systems typically do not allow adequate volume control or mixing, and are wasteful of scintillation cocktail. Pumps operating at the requisite low flow rates of 0.2 ml/min are frequently inaccurate and difficult to calibrate. There exists, therefore, a need for an automated system adaptable to a high pressure liquid chromatograph which provides for both the continuous or instantaneous identification of the radioactive components of the eluate and for the preservations of aliquots of the eluate for further analytical work with as little waste of materials as possible.